144 research outputs found
Reversible positioning of single molecules inside zero-mode waveguides
We have developed a hybrid nanopore/zero-mode waveguide device for single-molecule fluorescence and DNA sequencing applications. The device is a freestanding solid-state membrane with sub-5 nm nanopores that reversibly delivers individual biomolecules to the base of 70 nm diameter waveguides for interrogation. Rapid and reversible molecular loading is achieved by controlling the voltage across the device. Using this device we demonstrate protein and DNA loading with efficiency that is orders of magnitude higher than diffusion-based molecular loading.R21 HG006873 - NHGRI NIH HHS; R21-HG006873 - NHGRI NIH HHSPublished versio
Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing
Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.R01 HG009186 - NHGRI NIH HHS; R21 HG006873 - NHGRI NIH HHSAccepted manuscrip
Driven translocation of a semi-flexible polymer through a nanopore
We study the driven translocation of a semi-flexible polymer through a
nanopore by means of a modified version of the iso-flux tension propagation
theory (IFTP), and extensive molecular dynamics (MD) simulations. We show that
in contrast to fully flexible chains, for semi-flexible polymers with a finite
persistence length the {\it trans} side friction must be
explicitly taken into account to properly describe the translocation process.
In addition, the scaling of the end-to-end distance as a function of the
chain length must be known. To this end, we first derive a semi-analytic
scaling form for , which reproduces the limits of a rod, an ideal chain,
and an excluded volume chain in the appropriate limits. We then quantitatively
characterize the nature of the {\it trans} side friction based on MD
simulations of semi-flexible chains. Augmented with these two factors, the
modified IFTP theory shows that there are three main regimes for the scaling of
the average translocation time . In the stiff chain
(rod) limit , {}, which continuously
crosses over in the regime towards the ideal chain
behavior with {}, which is reached in the regime
. Finally, in the limit
the translocation exponent approaches its symptotic value , where
is the Flory exponent. Our results are in good agreement with available
simulations and experimental data
Sampling the proteome by emerging single-molecule and mass-spectrometry methods
Mammalian cells have about 30,000-fold more protein molecules than mRNA
molecules. This larger number of molecules and the associated larger dynamic
range have major implications in the development of proteomics technologies. We
examine these implications for both liquid chromatography-tandem mass
spectrometry (LC-MS/MS) and single-molecule counting and provide estimates on
how many molecules are routinely measured in proteomics experiments by
LC-MS/MS. We review strategies that have been helpful for counting billions of
protein molecules by LC-MS/MS and suggest that these strategies can benefit
single-molecule methods, especially in mitigating the challenges of the wide
dynamic range of the proteome. We also examine the theoretical possibilities
for scaling up single-molecule and mass spectrometry proteomics approaches to
quantifying the billions of protein molecules that make up the proteomes of our
cells.Comment: Recorded presentation: https://youtu.be/w0IOgJrrvN
Plasmonic nanopores for single-molecule detection and manipulation: towards sequencing applications
Solid-state nanopore-based sensors are promising platforms for next-generation sequencing technologies, featuring label-free single-molecule sensitivity, rapid detection, and low-cost manufacturing. In recent years, solid-state nanopores have been explored due to their miscellaneous fabrication methods and their use in a wide range of sensing applications. Here, we highlight a novel family of solid-state nanopores which have recently appeared, namely plasmonic nanopores. The use of plasmonic nanopores to engineer electromagnetic fields around a nanopore sensor allows for enhanced optical spectroscopies, local control over temperature, thermophoresis of molecules and ions to/from the sensor, and trapping of entities. This Mini Review offers a comprehensive understanding of the current state-of-the-art plasmonic nanopores for single-molecule detection and biomolecular sequencing applications and discusses the latest advances and future perspectives on plasmonic nanopore-based technologies
A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA
Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load
testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often
too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at
the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and
gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with
nucleic acid sequence based amplification (NASBA), this assay can detect concentrations of HIV RNA that match the clinically
relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and
requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample
preparation technologies to serve as an HIV viral load test for low-resource settings
Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors
Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules
Femtosecond photonic viral inactivation probed using solid-state nanopores
We report on detection of virus inactivation using femtosecond laser radiation by measuring the
conductance of a solid state nanopore designed for detecting single particles. Conventional methods
of assaying for viral inactivation based on plaque forming assays require 24–48 h for bacterial growth.
Nanopore conductance measurements provide information on morphological changes at a single
virion level.We show that analysis of a time series of nanopore conductance can quantify the detection
of inactivation, requiring only a few minutes from collection to analysis. Morphological changes were
verified by dynamic light scattering. Statistical analysis maximizing the information entropy provides
a measure of the log reduction value. This work provides a rapid method for assaying viral inactivation
with femtosecond lasers using solid-state nanopores.First author draf
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